1. Field of the Invention
The present invention relates to the use of electrorheology to form electrorheological crystalline masses of molecules, preferably macromolecules such as proteins, nucleic acids, carbohydrates, lipoproteins and viruses, through alignment of the molecule's permanent and/or induced dipole moments within a uniform electric field. The present invention also relates to the formation of electrorheological crystalline masses of molecules as a method for facilitating the formation of x-ray diffraction quality crystals of the molecules. One particular application of the present invention is the crystallization of proteins and their subsequent characterization by x-ray diffraction.
2. Description of Related Art
One of the fundamental problems faced by the biotechnology industry is a lack of knowledge regarding the three dimensional structures of the molecules being studied. X-ray crystallography is presently the only effective method known for determining the three dimensional structure of large molecules, referred to herein as macromolecules, such as proteins, nucleic acids, carbohydrates, lipoproteins and viruses, at a near atomic resolution level. X-ray crystallography works by exposing a crystal, i.e., a periodic lattice of like molecules, to x-rays. X-rays have a wavelength near 1.ANG. which is roughly equivalent to the interatomic spacings in crystals. As x-rays traverse a crystal, the x-rays are diffracted by the individual atoms in the crystal, thereby creating an x-ray diffraction pattern. The x-ray diffraction pattern can then be used to calculate the spatial relationships of the atoms of the molecule making up the crystal, i.e., the x-ray diffraction pattern can be used to determine the molecule's three dimensional structure.
In order to perform x-ray crystallography, one must first obtain an x-ray diffraction quality crystal. A diffraction quality crystal is a crystal whose periodic lattice has sufficiently few defects and distortions so as to create a consistent x-ray diffraction pattern, thereby enabling one to determine the three-dimensional structure of the molecule forming the crystal. The atomic resolution of the three dimensional structure calculated from an x-ray diffraction pattern depends on the number of distortions and defects in the crystal's periodic lattice.
Macromolecules, such as proteins, nucleic acids, carbohydrates, lipoproteins and viruses are frequently extremely difficult to crystallize. As a result, x-ray crystal structures for many macromolecules are either not available or extremely difficult to obtain. For example, most protein crystals initially form soft, nearly gelatinous lattices with a high solvent content and resulting distortions and defects. In order to improve the resolution-determining diffraction quality of the protein crystal, the scientist must continually modify the crystallization conditions such as solvent composition, temperature, salts, precipitants and pH, largely through trial and error. Crystallization thus is generally an iterative process which frequently takes many years before a diffraction quality crystal is produced.
The difficulty associated with generating x-ray diffraction quality crystals of macromolecules is evidenced by the fact that crystal structures have only been determined for about 1% of the more than 100,000 protein sequences that are presently known. Scientists thus are generally forced to study the physical properties of a molecule and to design compounds, such as drugs which recognize and bind to the molecule, without ever knowing the three dimensional structure of the molecule being studied. An improved method for generating x-ray diffraction quality crystals is therefore needed in order to enable the determination of the three dimensional structure of a greater number of macromolecules, particularly proteins.
Crystallization, in general, is the self-assembly of molecules into a periodic lattice. In order for the molecules to self-assemble into a crystal, several molecules must first come together with the proper orientation and initiate the periodic lattice. In order for the periodic lattice to grow, additional molecules must come into contact with the periodic lattice with the proper orientation. Because crystallization requires the self-assembly of molecules in an ordered fashion, there is a significant entropy barrier to crystallization. A method for reducing the entropy barrier to crystallization is needed. The present invention relates to the use of electrorheology to form highly ordered electrorheological crystalline masses of molecules which facilitate the formation of x-ray diffraction quality crystals of molecules.
Electrorheology is a phenomenon by which macroscopic particles dispersed within a dispersion fluid form an electrorheological solid when subjected to a uniform electric field.
In general, electrorheology involves the placement of a dispersion of macroscopic particles capable of forming an induced dipole within a uniform electric field. When the polarizable macroscopic particles are subjected to a uniform electric field, the positively charged protons inside the macroscopic particles are attracted to the negative pole of the uniform electrical field and the negatively charged electrons are attracted to the positive pole of the uniform electrical field. As a result, positive and negative charges are shifted within each macroscopic particle by the electric field, thereby causing the macroscopic particle to become an induced electric dipole.
When the macroscopic particles are subjected to a uniform electric field, the attractive and repulsive forces exerted on the macroscopic particles due to their induced dipole moments cause the macroscopic particles to line up relative to each other, positive dipole end to negative dipole end, forming chains of macroscopic particles. The chains of macroscopic particles quickly grow in length until the chain extends to the opposing ends of the chamber holding the electrorheological dispersion. Meanwhile, the chains combine to form chains of increasing thicknesses until an electrorheological solid of macroscopic particles is formed. Electrorheology was first described in U.S. Pat. No. 2,417,850 and has more recently been described in Halsey, et al. Scientific American (1993) 58-64, Chen, et al., Phys. Rev. Lett. (1992) 68:2555, and Block, et al., J. of Physics D: Applied Physics (1988) 21:1661-1677, each of which is incorporated herein by reference.
To date, electrorheology has only been performed on macroscopic particles having a diameter of between about 0.04-50 .mu.m. It is an object of the present invention to perform electrorheology on molecules, as opposed to macroscopic particles, in order to facilitate the crystallization of the molecules through the formation of an electrorheological crystalline mass of molecules.